Purpose: Compliance with
post-vasectomy semen analysis could be improved with the
availability of a simple, rapid and accurate home test.
SpermCheck® Vasectomy, a highly sensitive lateral flow
immunochromatographic diagnostic device, was designed to detect
extreme oligospermia or azoospermia in men after vasectomy. We
report the results of clinical and consumer testing of
SpermCheck® Vasectomy.

Results: A cohort of 144
post-vasectomy semen samples was tested in the clinical trial.
SpermCheck® Vasectomy was 96% accurate inpredicting
whether sperm counts were greater or less than a threshold of
250,000 sperm per ml, a level associated with little or no risk of
pregnancy. Sensitivity was 93% (95% CI 79% to 98%) and specificity
was 97% (91% to 99%). The positive predictive value of the test was
93% (79% to 98%), and most importantly the negative predictive
value was 97% (91% to 99%). The test gave a positive result 100% of
the time at sperm concentrations of 385,000/ml or greater. Consumer
studies with 109 lay volunteers showed that SpermCheck®
Vasectomy was easy to use. Volunteers obtained the correct or
expected test result in every case and the correct response rate on
a 20 question survey about the test was 97%.

Conclusions: SpermCheck®
Vasectomy, a simple and reliable immunodiagnostic test that can
provide evidence of vasectomy success or failure, offers a useful
alternative to improve compliance with post-vasectomy sperm
monitoring. It is currently the only Food and Drug Administration
approved test for this purpose.

Vasectomy is a safe and effective
method of male contraception. However, men who choose vasectomy
must continue to use other birth control methods until residual
sperm have been cleared from the reproductive tract. The time
necessary to achieve this clearance can be variable,1,2
and compliance with post-vasectomy semen analysis to confirm
azoospermia is poor.3-5 The inconvenience of
returning to the clinic and the embarrassment associated with
providing semen for analysis are cited as reasons for low patient
compliance.6 A diagnostic test that men could perform in
the privacy of their own home could lead to substantial improvement
in this area. Herr et al described an immunodiagnostic device
capable of detecting low numbers of sperm.7 This lateral
flow immunochromatographic test, SpermCheck® Vasectomy,
uses monoclonal antibodies that recognize the sperm specific
acrosomal protein SP-10.
SP-10 has been validated as a selective analyte for measuring
sperm concentrations. It is expressed only in the testis and is not
found in other organs.8 The SP-10 protein is present in
every male,9 is readily released from sperm with mild
detergents10 and remains soluble in aqueous solutions.
Thus, SP-10 is a postmeiotic, sperm specific biomarker amenable to
detection with monoclonal antibodies.11 A direct
relationship exists between sperm number and signal strength in
enzyme linked immunosorbent assays that measure SP-10
concentrations indicating that an immunodiagnostic device could be
developed using the SP-10 analyte.7In this report we
describe optimization, and clinical and consumer testing of
SpermCheck Vasectomy, a convenient, easy to use, rapid home test
useful for detecting azoospermia or extreme oligospermia in
post-vasectomy semen samples.

Materials And
Methods

Principle of the Assay

The SpermCheck® Vasectomy
test is a rapid qualitative test that detects the presence of sperm
in human semen at

concentrations of 250,000 sperm per ml
or greater. The test uses solid phase chromatographic immunoassay
technology. After liquefaction a volume of semen is mixed with a
buffer, applied to the device and the test result is read 7 minutes
later (fig. 1). The principle underlying operation of the
device is shown in figure 2. Representative test results and their
interpretation are described in figure 3.

FIG. 1. SpermCheck test procedure. After
liquefaction measured volume of semen is added to phosphate buffer
solution containing 0.1% Triton-X-100. If sperm are present
detergent releases SP-10 antigen (analyte) from sperm acrosome into
solution. When drops of solution are added to device well, if sperm
are present SP-10 antigen is available for antibody binding in test
pad and at test line on nitrocellulose membrane. Test line turns
reddish purple indicating positive result.

FIG. 2. Principle of operation of
SpermCheck immunochromatographic device. After mixing semen sample
with test solution 4 drops of mixture are dispensed into sample
well. Sample fluid migrates by capillarity through pads containing
lyophilized detector antibody to SP-10 protein. Detector antibody
is conjugated to gold particles. If sample contains sperm, upon
hydration of pad by applied sample complexes form consisting of
antibody-gold/SP-10 antigen. Immune complexes then flow up
nitrocellulose membrane. Membrane serves as solid support upon
which capture antibody (test band) and anti-species IgG antibodies
(control band) are immobilized. Immobilization of reagents on
nitrocellulose membrane in defined areas allows for formation of
distinct colored bands that can be read visually. Complex of
antibody-gold plus SP-10 antigen is captured by antibody on
membrane and reddish purple line appears at test band in device
window if sperm are present. If sample contains sperm below
detection level, no line appears at test band. Anti-species IgG
antibodies are immobilized at control band. Antibodies will bind to
excess (unbound) antibody-gold conjugates, resulting in formation
of distinct line at control band, telling user that device has
worked (flowed) properly. If control line does not appear within
designated incubation time, test is invalid and should be
repeated.

FIG. 3. Results of SpermCheck tests.
Diagnostic devices showing positive (A), negative
(B) and 2 types of invalid test results (C and
D). Control line must appear to insure result is
valid.

Rationale for
Sensitivity Level

The risk of pregnancy at low sperm
concentrations is a subject of intense debate. On the one hand
there are rare documented reports of confirmed paternity by men who
had multiple semen analyses negative for sperm before and after the
pregnancy.12 Conversely studies of couples presenting
with primary or secondary infertility show pregnancy rates of less
than 1 per 100 person-years of unprotected intercourse when the
sperm count is between 1 x 105 and 1 x
106/ml.13 Studies of candidate male hormonal
contraceptives have shown that suppression of sperm production to
severely oligospermic levels (less than 1 x 106/ml)
provides high contraceptive efficacy and that the failure rate is
proportional to the residual sperm count.14-17 On the
basis of these findings the consensus emerged that a sperm count
less than 1 x 106/ml is an appropriate target for
hormonal suppression of spermatogenesis.18 Taken
together these data support the concept that sperm concentrations
less than 250,000/ml are associated with little risk of pregnancy.
Although there are no consensus guidelines or uniform standards of
care for confirming sterility in men who underwent vasectomy, the
1 x 106/ml value has also been suggested as a
cutoff for post-vasectomy sterility.19 Given current
knowledge of the relationship between sperm counts following
vasectomy and fertility, and with the caveat that even repeatedly
documented azoospermia is not an absolute guarantee of sterility,
it was decided to develop the SpermCheck Vasectomy device with a
nominal detection limit of 250,000 sperm per ml.

Assay Development and Optimization

Semen samples were collected from healthy normal men and
vasectomized subjects. Sperm from normal donor semen were counted
using a Neubauer hemacytometer (Hausser Scientific, Horsham,
Pennsylvania) and pooled. Pooled seminal fluid from confirmed
azoospermic vasectomized donors was used to dilute the normal semen
to obtain test samples over a range of low sperm concentrations,
which were then used to determine the sensitivity of test lots of
SpermCheck Vasectomy devices. When parameters for manufacturing
devices with the desired sensitivity had been established, 2
production lots of 3,000 devices each were produced. Sensitivity
and precision studies confirmed that these devices gave positive or
negative results with semen samples ranging in sperm concentration
from 0 to 706,000/ml (data not shown).

Clinical Study Design

All studies involving human subjects
were reviewed and approved by institutional review boards at the
sites where patients were recruited and testing was performed. The
performance of SpermCheck® Vasectomy was compared to the
generally accepted standard procedure of determining sperm
concentration with a hemacytometer (the predicate device). The
study used patient samples obtained at various intervals after
vasectomy. To obtain approximately 25% semen samples with sperm
concentrations high enough to give positive test results, some
patients were asked to provide specimens at earlier times following
surgery than would normally be the case for post-vasectomy semen
analyses.

Patients who underwent vasectomy
who volunteered for the study were asked to return to the clinical
laboratory and provide a fresh semen sample as normally required by
their physician for routine followup after the procedure. They were
given the option of bringing 1 or more additional samples from the
second through sixth ejaculate after vasectomy. The patients
collected samples in private at the clinic or, if they preferred,
at home within 2 hours of presenting the sample. A physician or
technician in the physician’s office performed the SpermCheck test
with the patient’s sample by following the instructions provided in
the kit. A determination of sperm concentration was then made with
a hemacytometer following the standard operating procedures.
Technicians performing the hemacytometer counts were previously
trained in these procedures. This was a blinded study and the
person performing the hemacytometer count had no knowledge of the
results from the SpermCheck® Vasectomy test.

Standard Operating Procedure for Determining
Sperm Concentration

Samples with relatively abundant
spermatozoa were appropriately diluted with bicarbonate/formalin
buffer and 2 independent counts of at least 200 sperm each were
performed.20 Both chambers of an improved Neubauer
hemacytometer (Hausser Scientific, Horsham, Pennsylvania) were
scored and the average count recorded, provided that the difference
between the 2 counts did not exceed 5% of their sum (both values
were within 5% of the average). If the 2 counts were not within 5%
of their average, no result was recorded. The sample then was
remixed, and another hemacytometer was prepared and counted.

The number of 1 mm squares that were
counted to reach a minimum of 200 sperm varied depending on the
sperm concentration. To calculate the concentration of sperm in the
sample the average number of sperm in a 1 mm square was determined
by dividing the total number of sperm counted by the number of
squares counted. This quotient was multiplied by the dilution
factor, if any, and the result was multiplied by 104 to
give spermatozoa per ml.

Analysis of Semen Samples With Low Sperm
Concentrations

Semen samples with low sperm
concentrations were not diluted or centrifuged before counting. The
hemacytometer was placed in a humidified chamber for 10 minutes to
allow sperm to settle. Two independent counts were performed. A
minimum of 200 sperm were counted on each side of the hemacytometer
and the 2 counts averaged. If the 2 counts did not agree within 5%
of their mean, results were not recorded, a new hemacytometer was
prepared and 2 independent counts were performed again.

Analysis of Semen Samples With Sperm
Concentrations Less Than 111,000/ml

If fewer than 200 sperm were counted
in all 18, 1 mm squares on both sides of a hemacytometer, then the
sperm concentration was less than 111,000/ml. In such cases 5
separate hemacytometers were prepared and counted. The count for
each side (9 squares) of all 5 hemacytometers (90 squares total)
was used to calculate the mean sperm concentration with 95%
confidence intervals.

Consumer Study Design

Two consumer studies were performed.
In the first study a total of 51 subjects were enrolled, 31 who had
undergone vasectomy and 20 who had not. A total of 50 men completed
the study (1 who had a vasectomy withdrew from the study before
completing the testing). Participants ranged in age from 18 to
older than 65 years and in education from high school graduates to
holders of advanced academic and professional degrees, and were
employed in a range of occupations.

Subjects were provided a test kit with
printed instructions. Subjects performed the test at home with
their own semen sample, recorded the test result, and answered
questionnaires about the test, instructions and results (see
Appendix). The subjects then returned the questionnaire and the
remainder of the semen sample to the laboratory where the
SpermCheck® Vasectomy test was repeated and a
hemacytometer count performed. Thus, this study compared lay vs
professional users and also compared SpermCheck to the predicate
device.

In the second consumer study
volunteers were provided with semen samples at known sperm
concentrations along with test kits and instructions. Subjects
performed the SpermCheck test at the study site without assistance,
recorded the results and answered a questionnaire. This group of 37
nonnative and 16 native English speaking subjects (17 to 67 years
old) included 10 high school graduates, 29 college graduates and 13
with advanced degrees. Of the participants 10 were women. The test
samples for this study were prepared by diluting a pool of semen
from normal donors with semen from azoospermic vasectomized donors
to obtain sperm concentrations of 17,000, 350,000 and
1,000,000/ml.

Results

Clinical Trial

The results of SpermCheck®
Vasectomy testing and hemacytometer counting of 144 semen samples
provided by 100 patients who underwent vasectomy are depicted in
figures 4 and 5. A summary of the data is provided in tables 1 and
2. Of the 40 semen samples with sperm concentrations greater than
250,000/ml 37 tested positive for the presence of sperm with the
device (93% sensitivity). Of the 104 samples with sperm
concentrations less than 250,000/ml 101 tested negative (97%
specificity). The overall accuracy of SpermCheck in predicting
whether the sperm concentration determined by hemacytometer
counting was greater or less than the threshold value of 250,000/ml
was 96%. The PPV of the test was 93% and the NPV was 97%.

Consumer Testing

There was 100% agreement between the SpermCheck®
Vasectomy results obtained by 50 home users and those obtained by
laboratory personnel retesting the same semen samples. There was 1
disagreement between the Sperm- Check Vasectomy results and the
hemacytometer counts among the 50 tests (98% agreement). Each of
the 59 participants in a second consumer study tested 3 prepared,
coded semen samples for a blind study. All subjects obtained the
expected SpermCheck® Vasectomy test result with each
sample.
The participants in both consumer studies completed the same
questionnaire after performing the SpermCheck® Vasectomy
test (see Appendix). The results are presented in table 3, and
indicate that users found the test easy to perform and were able to
understand the instructions, correctly reading and interpreting
their test result.

Discussion

SpermCheck® Vasectomy was
highly accurate in predicting whether sperm count was greater or
less than 250,000/ml. Most of the few discordant results observed
were within a narrow range of sperm concentrations and could be due
to errors in sperm counting rather than diagnostic device failures.
Of the 6 discrepant samples 5 had sperm concentrations close to the
cutoff level of 250,000 sperm per ml. Two false-positive results
were observed with sperm concentrations of 235,000 and 246,389
sperm per ml, while 3 falsenegative results were observed with
sperm concentrations of 296,667, 365,556 and 384,722 sperm per ml.
Determination of low sperm counts is notoriously inaccurate even
with a rigorous counting protocol such as that used in this study.
When we consider the inherent error rates of hemacytometer counting
at these low sperm concentrations, it is difficult to state with
certainty that any of these discrepant Sperm-Check results were
truly incorrect.20,21 One other falsepositive sample had
a low sperm concentration (667 sperm per ml).

Sensitivity (and, thus,
negative predictive value) is the most important parameter to
consider in evaluating the performance of SpermCheck®
Vasectomy since a false-negative test could potentially lead to an
unwanted pregnancy (the only consequence of a false-positive result
is the need for retesting at a later date). The highest sperm
concentration of any semen sample that gave a negative SpermCheck
result was 384,722/ml according to the hemacytometer count. Thus,
within this cohort a negative result indicated that the sperm
concentration was no higher than approximately 385,000/ml. Stated
another way although the device shows some variability around the
250,000 sperm per ml cutoff the sensitivity of the device is such
that one can predict a positive result 100% of the time when sperm
concentrations are 385,000/ml or greater. Given that 385,000/ml is
well below the 1 million per ml level targeted in many studies of
male contraceptives, the SpermCheck® Vasectomy device
may also be of value in measuring the decline into oligospermia in
trials of male contraceptives.

The data from 2 separate
consumer studies with a diverse cohort of more than 100 volunteers
showed that lay users can correctly follow the instructions, obtain
expected test results and correctly interpret the meaning of these
results when using SpermCheck® Vasectomy. One question
concerning the importance of communicating test results to the
physician caused some confusion, with 17% incorrect responses. Test
instructions were modified to clarify and emphasize this point. The
sole discordant test result in the home use study involved a semen
sample from a man who had undergone vasectomy 2 years earlier. Both
the study subject and the professional user obtained a presumptive
false-positive SpermCheck result with this sample. Interestingly
the semen sample was not azoospermic but had a sperm concentration
of 7,000/ml by hemacytometer counting. These few sperm were not
motile. A subsequent semen sample from the same donor was negative
by SpermCheck and hemacytometer. Similar intermittent reappearance
of sperm late after vasectomy has been reported by
others.12

It is important to note the
lack of a true standard of care or even clear and uniform
guidelines regarding what constitutes acceptable evidence for
declaring a vasectomy successful. It is not within the scope of
this report to debate the merits of various standards for
post-vasectomy semen analysis, nor is it our intent to suggest that
a single SpermCheck® Vasectomy test, in and of itself,
represents a new standard for clearing patients to discontinue
other methods of contraception following vasectomy. While
SpermCheck cannot provide proof of sterility it is clear from the
literature that microscopic sperm counting does not constitute such
proof either. Unfortunately many patients apparently decide that
their procedures were successful without any evidence and without
adequate consultation. In this context we believe that
SpermCheck® Vasectomy can have a role in improving
compliance, and improving communication between patients and their
physicians.

FIG. 4. Summary of clinical testing of
SpermCheck Vasectomy. Total of 101 men were recruited consecutively
from among patients seeking vasectomy at 2 clinics between May 2006
and February 2007. One participant was lost to followup before
providing any semen samples for analysis, leaving 100 subjects
enrolled in trial. Men donated total of 145 semen samples for
testing but 1 result was excluded from analysis since SpermCheck
test was not performed correctly. Of 145 semen samples tested 144
were included in analysis, 60 early post-vasectomy samples and 84
regular clinical followup samples.

FIG. 5. Plot
of sperm concentrations by test result with horizontal line at
concentration of 250,000/ml

Conclusions

SpermCheck® Vasectomy is a
fast, simple, sensitive and reliable home test that can provide
patients with useful information about sperm counts when used at
appropriate times after surgery. It may also be used at longer
intervals to give assurance against late recanalization or to
confirm recanalization when pregnancy has occurred after vasectomy
was considered successful. SpermCheck® Vasectomy may
prove to be a useful adjunct to current practice in post-vasectomy
sperm detection, and the availability of this convenient and
economical home testing option could improve patient compliance.
The Food and Drug Administration approved this device for
qualitative detection of low concentrations of sperm following
vasectomy in January 2008.

Please indicate your agreement
with each statement below using a 1–5 scale, where
1 =strongly disagree and 5 =strongly agree.

1. The test instructions were easy to
understand. ( )

2. It was easy to follow the
instructions. ( )

3. The answers to frequently asked
questions were helpful. ( )

4. It was easy to add the semen to the
SpermCheck Solution Bottle. ( )

5. It was easy to apply the mixed
sample to the test device. ( )

6. Reading the test result was easy. (
)

7. The control line was clearly
present and easy to read. ( )

8. The instructions clearly explain
what to do if a test result is invalid. ( )

The following are multiple
choice questions about performing the test. Please circle the
letter of the best answer to each question. Please mark only one
answer for each question. You may refer to the package insert at
any time as you answer these questions.

9. After collecting your semen sample,
how long should you wait before adding it to the SpermCheck
Solution Bottle?

a. 15 to 30 minutes

b. at least 10 minutes

c. at least 20 minutes, but not more
than 3 hours

d. you should start the test
immediately

10. When you remove semen from the
Semen Collection Tube to add to the SpermCheck Solution Bottle, it
is important to

a. use the dropper to mix the semen
sample

b. avoid solid or sticky material

c. avoid air bubbles

d. do all of the above

11. How much semen should you add to
the SpermCheck Solution Bottle?

a. fill the dropper to the bottom
line

b. fill the dropper to the middle
line

c. fill the dropper to the top
line

d. you should add all of the semen
sample to the bottle

12. After mixing the semen with the
SpermCheck Solution, how long should you wait before adding the
sample to the sample well of the test device?

a. 1 minute

b. 2 minutes

c. 5 minutes

d. 20 minutes

13. How many drops of the
semen/solution mixture should you add from the SpermCheck Solution
Bottle to the sample well of the test device?

a. 1 drop

b. 2 drops

c. 4 drops

d. 8 drops

14. When should you read the test
result?

a. no more than 7 minutes after adding
the sample to the device

b. exactly 7 minutes after adding the
sample to the device

c. at least 7 minutes after adding the
sample to the device

d. as soon as you see the control
line

The following are true or
false questions about reading your test and understanding the
results. Please mark (T) for true and (F) for false statements. You
may refer to the package insert at any time as you answer these
questions.

15. If the test result is
positive, other birth control methods should still
be used. ( )

16. An invalid test
means the result is negative. ( )

17. A weak signal at the test
line position with a clear control line is a
negative result. ( )

18. If the test result is
negative the sperm count is very low and the risk
of pregnancy is extremely small. ( )

19. If the test result is
positive you should wait two weeks and test a new
sample. ( )

20. If your result was negative, you should
talk to your doctor about your decision to stop using other birth
control. ( ).

Abbreviations and Acronyms

NPV = negative predictive
value

PPV = positive predictive
value

SP-10 = sperm protein 10 [ARCV1 gene product]

References

1. Alderman PM: General and anomalous
sperm disappearance characteristics found in a large vasectomy
series. Fertil Steril 1989; 51: 859.

2. Barone MA, Nazerali H, Cortes M,
Chen-Mok M, Pollack AE and Sokal D: A prospective study of time and
number of ejaculations to azoospermia after vasectomy by ligation
and excision. J Urol 2003; 170: 892.